Epithelial brush border membrane (BBM) Na+/H+-exchanger 3 (NHE3) is a critical transporter responsible for intestinal and renal neutral Na absorption. In small intestine, NHE3 inhibition is a major mechanism for most diarrheal diseases. Therefore understanding the regulatory mechanism of NHE3 is of great significance in gastroenterology. This proposal will examine the roles of lipid rafts and heat shock cognate protein 70 (Hsc70) in the regulation of NHE3. The hypothesis to be tested is that 1), lipid rafts are involved in NHE3 membrane partitioning and trafficking and are required for the proper function of NHE3; and 2), through interaction with NHE3 and endocytic machinery Hsc70 is involved in basal and regulated NHE3 activity by facilitating NHE3 trafficking and/or NHE3 complex assembly/disassembly. This hypothesis is supported by strong preliminary data (i), NHE3 is present in the lipid rafts of both rabbit ileal BBM and cultured opossum kidney (OK) cells; (ii), EGF stimulates NHE3 activity by increasing NHE3 amount in BBM lipid rafts; (iii), Disruption of lipid rafts inhibits endocytosis of ileal BBM NHE3 and the NHE3 activity of OK cells; (iv), NHE3 exists as large complexes (400-900 kDa) in Caco-2/E3V (Caco-2 cells stable transfected with NHE3 tagged with VSVG) and kidney proximal tubule cell line OK/E3V. NHE3 complexes contain up to 10 distinct proteins, including Hsc70 in PS 120/E3V cells, Caco-2 cells and kidney proximal tubule epithelial cells; (v), Co-immunoprecipitation and in vitro binding assay show that Hsc70 interacts with NHE3 directly. The proposed studies will lead to the understanding of two novel mechanisms by which epithelial NHE3 is regulated: compartmentation in lipid rafts and complex formation with Hsc70.